Novel antifungal antibiotic substance, process for production of the same, and agricultural and horticultural fungicidal composition containing said substance

ABSTRACT

The antifungal antibiotic substance F-1028 having the following formula,   OR AN ACID SALT THEREOF, A PROCESS FOR THE PRODUCTION OF THE SAME BY FERMENTATION, AND A FUGICIDAL COMPOSITION USEFUL FOR AGRICULTURAL AND HORTICULTURAL USE.

United States Patent Hata et al. Sept. 30, 1975 NOVEL ANTIFUNGAL ANTIBIOTIC [56] References Cited SUBSTANCE, PROCESS FOR PRODUCTION UN STATES PATENTS OF THE SAME, AND AGRICULTURAL AND 3 687 926 8/1972 HORTICULTURAL FUNGICIDAL COMPOSITION CONTAINING SAID SUBSTANCE Inventors: Toju Hata; SatoshiOmura; Michiko Katagiri, all of Tokyo; Juichi Awaya, Soka; Shimpei Kuyama, Tokyo; Shizuo Higashikawa, Kunitachi; Kazuomi Yasui; l-Iaruko Terada, both of Hino, all of Japan Assignees: Nihon Tokushu Noyaku Seizo Kabushiki Kaisha, Tokyo, Japan; The Kitasato Institute, Tokyo, Japan Filed: Mar. 20, 1974 Appl. No.: 453,040

Related US. Application Data Division of Ser. No 272,217, June 17, 1972.

Foreign Application Priority Data June 29, 1971 Japan 46-47411 US. Cl. 195/80 R; 260/210 AB; 260/21 1 Int. Cl. C12D 9/00 Field of Search 195/80 Arima et al 195/80 X Primary ExaminerLionel M. Shapiro Assistant ExaminerRobert J. Warden Attorney, Agent, or FirmSherman & Shalloway [57] ABSTRACT The antifungal antibiotic substance F-l028 having the CH3CHCONHCH or an acid salt. thereof, a process for the production of the same by fermentation, and a fugicidal composition useful for agricultural and horticultural use.

5 Claims, 2 Drawing Figures US. Patent TRANSMISSION RATIO (70) Sept. 30,1975 3,909,361

Fig.l uv SPECTRUM OF AQUEOUS SOLUTION OF ANTIBIOTIC SUBSTANCE. F-IO28 HYDROCHLORIDE g 0.6 5 E 04 2 0.2 LU

l l l 1300! l l L LENGTH OF WAVE (my) Fig,2 IR SPECTRUM OF MIXTURE 0F F-IO28 HYDROCHLORIDE WITH POTASSIUM BROMIDE I l I I I l I I l l I l I I l I l I J l 4000 2800 I500 I000 500 NUMBER OF WAVE (cm-I NOVEL ANTIFUNGAL ANTIBIOTIC SUBSTANCE, PROCESS FOR PRODUCTION OF THE SAME, AND AGRICULTURAL AND HORTICULTURAL FUNGICIDAL COMPOSITION CONTAINING SAID SUBSTANCE This is a division of application Ser. No. 272,217, filed July 17, 1972.

This invention relates to a novel antifungal antibiotic substance F-l028 and an acid salt thereof, a process for producing the same by fermentation, and a fungicidal composition useful for agricultural and horticultural use.

We have now found that an antifungal antibiotic substance hitherto unknown can be produced by cultivating F-l028 substance-producing strain belonging to the genus Streptomyces in a culture medium containing a carbon source, a nitrogen 'source and a mineral under aerobic conditions.

Before this invention, the existence of a strain belonging to genus Streptomyces which can produce substance F-l028 having strong antifungal action and growth inhibiting action on various fungi, especially molds, which cause plant diseases was not known.

The novel antifungal antibiotic substance named F- 1028 substance exhibits strong fungicidal activity against various fungi which cause plant diseases but only weak antibacterial action on various bacteria.

This F-l028 substance has the following molecular formula and has the following properties (i) (ix);

i. it has a molecular weight of 219,

ii. it is colorless crystal, v

iii. the melting point of the hydrochloric acid salt thereof is above 195C (decomposition) iv. the specific rotation of the hydrochloric acid salt thereof is [01],, 63.2 (c=l, H O),

v. the UV spectrum of an aqueous solution of the hydrochloric acid salt thereof has no peculiar absorption as shown in FIG. 1.

vi. the IR spectrum of a mixture of the hydrochloric acid salt thereof with potassium bromide is as shown in FIG. 2,

vii. it has Rf value in thin-layer chromatography with silica gel of 0.68 with a developing solvent consisting of n-propanol-pyridine-acetic acid-water in a ratio of :10:33: 1 2; and 0.21 with a developing solvent consisting of butanol acetic acid-water in a ratio of 3:1:2,

viii. it is readily soluble in water and insoluble in chloroform, benzene and ether, and

ix. the color reaction is positive with ninhydrin, Ehrlich, Elson-Morgan, Tollens and Benedict reagents, and negative with Molisch, Sakaguchi, maltol and ferric chloride reagents.

This F-lO28 substance has activity against Archimycetes, Phycomycetes, Ascomycetes, Basia'iomycetes, and Fungi imperfecti. It can be used for example as a plant-protecting fungicide having both preventive and curative effects against a wide variety of plant pathogens causing such diseases as blast, sheath blight, bacterial leaf blight, or helminthosporium leaf spot of rice, leaf rust of wheat,v bacterial soft rot of Chinese cabbage, brown rot of peach, leaf spot of banana, gray mold of strawberry and other crops, downy mildew of grape, anthracnose of grape, apple and pear, stem rot of vegetables, anthracnose of melons and cucumber, melanose of citrus fruits, powdery mildew of wheat, apple, and cucumber, fungi causing black spot, for example, brown spot disease on apple or early blight on potato, fungi causing scab, for example, scab on pear or apple.

The present inventors newly separated the F-l028 substance-producing strain from soil and named it Streptomyces kagawaensis.

The Streptomyces kagawaensis nov. sp. (FERM-P No. 953: ATCC No.2l8l 1) is characterized by the following microbiological properties.

I. Morphological properties:

According to the microscopic observation, the aerial mycelium on a synthetic agar medium and a protein agar medium is branched irregularly. Sporangiopere forms closed spirals with no whorl. Spore is ellipse in size of 0.7,u, X 0.91.1. and has spinous surface.

11. Cultural characteristics on various culture media:

1. Sucrose-nitrate agar medium (at 27C) Growth; Good, pale yellowish brown to light pale yellow Aerial mycelium; Powdery, abundant, grayish brown Reverse; Yellow Soluble pigment; Pale brown 2. Glucose-asparagine agar medium (at 27C) Growth; Good, pale yellow to pale yellowish brown Aerial mycelium; Powdery, abundant, pale pink to brownish white Reverse; Yellowish brown Soluble pigment; Pale yellow 3. Glycerine-asparagine agar medium (at 27C) Growth; Good, yellowish brown, vegetative mycelium enters into the medium Aerial mycelium; White to pinkish white Reverse; Brown Soluble pigment; Yellowish brown 4. Starch-inorganic salt agar medium (at 27C) Growth; Good, pale yellow Aerial mycelium; Powdery, brownish white to pale brown Reverse; Yellowish brown Soluble pigment; Yellowish brown 5. Thyrosine agar medium (at 27C) Growth; Light yellowish brown to dark brown Aerial mycelium; White to brownish white Reverse; Brown to dark brown Soluble pigment; Black 6. Nutrition agar medium (at 27C) Growth; Light yellowish brown Aerial mycelium; None Reverse; Brown Soluble pigment; Yellowish brown 7. Yeast-malt agar medium (at 27C) Growth; Good, yellowish brown, vegetative mycelium enters into the medium Aerial mycelium; Cotton like, white to pale pink Reverse; Brown Soluble pigment; Yellowish brown 8. Oat meal agar medium (at 27C) Growth; Good, yellowish brown, vegetative mycelium enters into the medium Aerial mycelium; Powdery,

brown Reverse; Brown Soluble pigment; Yellowish brown 9. Peptone-yeast iron agar medium (at 27C) Growth; Lustrous yellowish white Aerial mycelium; None Reverse; Grayish brown Soluble pigment; Dark brown 10. Loeffler coagulated serum agar medium (at 27C) Growth; Lustrous brown Aerial mycelium; Scant, white Soluble pigment; Brown, no liquefaction l 1. Egg agar medium (at 27C) Growth; Creased, yellowish brown to dark brown Aerial mycelium; White to dim white Reverse; Brownish brown Soluble pigment; Dark yellowish brown, no liquefaction 12. Potato plug (at 27C) Growth; Thick, raised, lustrous yellowish brown Aerial mycelium; Scant, white Soluble pigment; Yellowish brown to dark brown 13. Carrot plug Growth; Thick, raised, lustrous yellowish brown Aerial mycelium; Cotton like, white Soluble pigment; Scant, pale yellowish brown 14. Glucose-peptone gelatin medium (at 27C) Growth; Creased, pale grayish brown Aerial mycelium; Pale grayish white 7 Soluble pigment; Brown 15. Skim milk medium (at 27C) Growth; Ring or membrane, pale yellowish brown Aerial mycelium; Scant, white Soluble pigment; Pale yellowish brown III. Physiological properties:

1. Optimum conditions for growth:

Temperature: between 25 to 30C pH: between 6 to 8 Aerobic condition 2. Growable conditions: Temperature: between 15 to 38C pH: between 4 to 9 Formation of melanine: positive Reduction of nitric acid: weak Milk coagulation: weak Milk-peptonization: moderate Gelatin-liquefaction: weak Decomposition of starch: positive Formation of hydrogen sulfide: positive 10. Dissolution of coagulated serum: negative IV. Assimilation of carbon sources:

The assimilation of carbon sources was tested by the method of D. G. Pridham et al.

Good assimilation:

Arabinose, glucose, galactose, glycerol, levulose, mannose, maltose, melibiose, sucrose, trehalose, inositol, mannitol.

abundant, bright 0 Moderate assimilation:

Lactose, xylose.

o Slight assimilation:

Raffinose, rhamnose, salicin, inulin, sorbinol.

o No assimilation:

Melezitose, sorbose, adonitol, dulcitol.

According to these microbiological properties, the F-l028 substance-producing strain, which grows with pale pink or grayish brown aerial mycelium and produces a brown or yellowish brown soluble pigment on a protein agar medium, is considered to belong to Strepromyces, particularly Streptomyces lavendulae.

Among known species of Streptomyces having these characteristic properties, Streptomyces lavendulae, Streptomyces venezuelae and Streptomyces verginiae, described in Bergeys Mannual of Determinative Bacteriology, 7th ed. and the Actimomycetes by Waksman, Vol. 2, are similar to the strain of this invention.

However, the spore surfaces of these three strains are smooth, but on the contrary, that of the F-l028 substance-producing strain is spinous. Comparison of the cultural properties between the F-l028 substanceproducing strain and these three strains shows that the three strains produce no soluble pigment but the F- 1028 substance-producing strain produces yellowish brown soluble pigment on sucrose-nitrate agar medium. Also, in the assimilation of carbon sources, any of these three strains shows little or no assimilation of merbiose, sucrose, inositol and mannitol, but the F- 1028 substance-producing strain assimilates these saccharides well.

By the differences of the morphological, cultural and physiological properties, the F-l028 substanceproducing strain can be clearly distinguished from Streptomyces lavendulae, Streptomyces venezuelae and Streptomyces verginia. The present inventors classified the F-l028 substance-producing strain as Streptomyces Iavendulae group in view of the-cultural and physiological properties, but distinguished it as a new strain from the known microorganisms under the name of Streptomyces kagawaensis nov. sp., according to the differences of spore surface structure and saccharide-assimilation and the possibility of producing a new antibiotic F-1028 substance.

Streptomyces kagawaensis is easily converted to a mutant strain by means of artificial mutations such as application of an ultraviolet ray, X-rays, or a high frequency wave, or radial rays, chemicals.

In the process of the present invention, there are employed all strains, which have a productivity of F-l028 substance, including mutants of a standard strain which are not clearly distinguished from Streptomyces kagawaensis as a standard strain.

The important aspect of the process of the present invention is based on the steps of culturing the F- 1028 substance-producing strain belonging to Streptomyces kagawaensis and isolating the antibiotic F-lO28 substance from the culture medium.

In this invention, the F-l028 substance-producing strain is cultured in a culture medium containing nutrients utilized by general microorganisms. As the nutrient source, all of the known nutrients for actinomyces can be used, for example, commercially available glucose, starch, glycerol, sucrose maltose, dextrin, molasses and fatts as carbon sources (shown in Table I) will now be described.

Examples of the nitrogen source are' commercially available soybean flour, meat extract, peptone, yeast, corn steep liquor, powdered cotton seed, powdered peanut, N-Z amine, casein, ammonium sulfate, ammonium nitrate and sodium nitrate (shown in Table 2).

Examples of the minerals (or inorganic salts) are calcium carbonate, sodium chloride, potassium chloride, phosphate, magnesium sulfate. If desired, a trace amount of metallic salts can be incorporated.

Any substance of these nutrients may be used, if it is assimilated by the F-l028 substance-producing strain and useful for producing the F-l028 substance.

Production of the F-l028 substance in a culture medium containing various carbon sources will now be described. I

ln a test tube (20 mm in diameter) was placed 13 ml of a liquid culture medium containing a basic medium, which consisted of peptone 0.5 meat extract 0.5 dried yeast 0.3 sodium chloride 0.5 and calcium carbonate 0.3 and the carbon sources described in the following table (shown in Table I). Then the seed culture of the F-l028 substance-producing strain was inoculated to the culture medium. The inoculated test tube was incubated with shaking in a reciprocated shaker at 27C. The production of the F-l028 substance is shown in Table I.

Table 1 Two days Three days F4028 F-l028 Carbon source pH mcg/ml pH meg/ml l Glucose 2.0 7c 7.4 632 7.6 280 2 Glycerol 2.0 7.4 452 7.4 258 3 Starch 2.0 72 7.6 548 8.0 148 4 Glucose l .071

Starch L 70 7.4 538 8.0 184 5 Glycerol L07:

Starch 1.0% 7.6 590 8.0 2l0 6 Maltose 2.0 8.2 78 8.4 32 7 Dextrin 2.0 71 7.8 284 7.8 420 8 Sucrose 2.0 it 8.4 36 8.4

Production of the F -l028 substance in a culture medium containing various nitrogen sources will now be described.

To a basic medium consisting of glucose 2.0 sodium chloride 0.5%, calcium carbonate 0.3 the nitrogen sources as shown in the following table were added. The culture medium was adjusted to pH 7, sterilized, inoculated with'the seed culture of the F4028 substance-producing strain and shaken at 27C. The production of the F-l028'substance is described in Table 2.

The best cultural method uses a liquid culture, expecially a submerged tank culture with aeration at temperatures ranging over the extent in which the F-l028 substance-producing strain is growable and produces the F-l028 substance, preferably at 25 to 35C. The practical culture is conducted at about 27C and continued until the F-l028 substance is sufficiently accumulateduThe pH value of the cultural medium is usually=frorn 6 to 9, preferably 6.2- to 8.5, especially 65 to 8. The concentration of the F-l028 substance in the culture medium becomes maximum within 2 to 5 days by. both shaking culture and tank culture method. However, since the days needed toget the maximum concentrationof the 5-1028 substance may be varied in accordance with condition of aeration and agitation in the same culture medium at the same temperature, it

is desirable todetermine the potency of the F-l028 substance. When the potency is maximum, the culture is stopped and the F-l028 substance is extracted. To determine the potency of the F-l028 substance, there was employed a method of measuring an inhibition circle by using Sclerotinia cinerea (hereinafter referred to as Sc) in accordance with a conventional way.

The practice was conducted as follows. One Kgof potato and 15 lit. of tap water were boiled for 30 minutes and the potatos were filtered away by gauze. Then to the filtrate, water was added in an amount of 5 lit. and commercially available sucrose was incorporated at 2% to prepare a culture medium for Sc. To this liquid medium agar was added at 1.5 and it was placed a test tube in order to make a slant culture medium. Sclerotinia cinerea strain wasinoculated on the slant medium and incubated for 4 to 7 days at 27C and 2 or 3 loopful of Sc strain on the slant culture medium were inoculated in ml of Se culture, medium containing agar at 0.3 in a Sakaguchis flask. After the flask was shaken for 4 days at 27C, the cultivated medium was homogenized for 2 minutes at 9,000 r.'p.m. It was used then for seeding. Thereafter, 10 ml of the Sc liquid medium added agar at l to 1.2 was placed in a petri dish, solidified and covered with 4 ml of the same agar medium mixed with the cultivated medium at 10 This plate culture medium was used in a conventional disc method. The culture was continued for 2 days at 27C. In this method, a 200 meg/ml solution of the pure F-l028 substance forms aninhibition zone with about 35 mm diameter. A linear relationship'between the concentration of the antibiotic and the diameters of inhibition zones stands at a range 10 to 200 meg/ml.

Since the F-1028 substance-has physical and chemivcalproperties as described hereinafter, the substance can be extracted and purified in accordance with the properties. The F-l028 substance was produced in the culture liquid, the mycelia are removed away by methods of centrifuging or filtration with-auxiliary filters such as kieselguhr at acidity or neutrality. Then the F 1028 substance was adsorbed on a cation exchange .resin, for example lRC-50(Na or IR-l20(l-l by the .batch or column method, and eluted with acidic or basic water. The F-l028 substance is a strongv basic compound which is not substantially adsorbed on IRC- 50(H but it is adsorbed on the resin in Na type. For instance, the filtrate of the fermentation mixture is treated with oxalic acid, and adsorbed on IRC- 50(Na and the antibiotic was'eluted rapidly with 1 N-HCl. In the case of using activated carbon, the.- F- 1028 substance can be adsorbed on the carbon and eluted with aqueous acetone and aqueous methanol but not substantially.

After the eluate containing the F-l028 substance was concentrated under reduced pressure, the concentrate, if necessary, was desalted with methanol, ethanol and an excessive amount of methanol, ethanol and acetone was added to precipitate the F-l028 substance in the form of powder. The eluate from the resin decomposed easily with coloring in brown by drying directly under reduced pressure. It was necessary to treat the F-l028 substance rapidly, because its activity became lower and it coloured in yellow when it was allowed to stand for a long time. The mixture containing the F- 1028 substance was decomposed in brown by precipitation with methanol, ethanol and acetone and drying. However, the F-l028 substance is very stable as far as it is in the form of aqueous solution. The crude powder obtained was purified by methods such as carbon chromatography or Sephadex chromatography to obtain a' white powder.

The eluate as hydrochloride was then passed through a column of IR-4B resin to be converted to a free type and thereafter to a sulfate by eluting with sulfuric acid. In this case, if the amount if IR-4B resin was not sufficient and the eluates from IR-4B resin were passed through a column of Sephadex G-lO, two fractions were eluted from the Sephadex column. The first fraction was a sulfate of the F-1028 substance and the second was a hydrochloride. The latter fraction was again passed through the column of IR-4B resin and eluted with sulfuric acid and the eluate was subjected to the Sephadex column to form a sulfate. The F-1028 substance isolated and purified by the methods described above, has the following more detailed physical and chemical properties.

1. Colorless needle-like or plate-like crystal 2. Melting point (hydrochloride), decomposed at above 195C 3. Elemental analysis (as hydrochloride) Found: C: 30.94%, H: 6.58%, N:

25.91%, Cl: 23.18% 4. Molecular formula: C H N O (molecular weight: 219, from elemental analysis,

titration and NMR spectrum) 5. Equivalent: 122.5 (from titration) 6. Specific rotation (hydrochloride) 7. PKa: 7.02 and 8.16 8. Ultraviolet absorption spectrum:

No peculiar absorption as shown in FIG. 1 9. Infrared absorption spectrum:

As shown in FIG. 2

l0. Rf value:

When a thin layer chromatography based on silica gel is used with a developing solvent consisting of butanol, acetic acid and water in a ratio 3: 1:2, Rf value is 0.25. When the solvent consists of npropanol, pyridine, acetic acid and water in a ratio of :10:3:12, Rf value is 0.68.

l l. Solubility:

Readily soluble in water;

Soluble in methanol and dimethyl sulfoxide;

sparingly soluble in ethanol;

Insoluble in chloroform, benzene and ether. 12. Color reaction:

Positive: Ninhydrin, Ehrlich, Elson-Morgan, Tollens,

and Benedict reactions Negative: Molisch, Sakaguchi, maltol, and ferric chloride reactions 13. It retains its activity in an acidic solution, but the activity is reduced when the solution becomes neutral to alkaline. Biological properties of the F-1028 substance 1. Antimicrobial activity:

The minimum inhibitory concentration (MIC) of the F-1028 substance for various microorganisms was examined by the agar dilution method, and the results are given in Table 3.

Table 3 24 hours 48 hours Microorganism mcg/ml mcg/ml Staphylococcus aureus FDA 209 p 100 Surcina lulea PCI 1001 3.12 3.12 Bacillus sublilis PCI 219 100 100 Mycobaclerium ATCC 607 100 100 Escherichia cali NIHJ 100 100 Vibrio comma 904 12.5 100 48 hours 72 hours Candida albicans 100 100 Sacchammyces cerevisiae 100 100 Holmodendrum pedrosei 25 25 Trichaphylan rubrum 100 100 ()phiubolus miyabianus 25 25 Aspergillus niger 100 100 Xanthomanas oryzae 50 100 Xunlhomonas cilri 100 100 Alrernuria japonica 50 50 Alternaria kikuchiana 100 100 Sclerun'nia cinerea 12.5 25 Sclerolinia scleroliorum 1.56 3.12 Bomrylis fabae 6.25 25 Bolorytis cinerea 6.25 12.5

Toxicity (i) No abnormality Mice (intravenous injection) mg/kg (oral administration) 500 mg/kg Young carp: no trouble with 250 ppm (ii) Mice (intravenous injection) (oral administration) LD mg/kg LD 750 mg/kg Accordingly, the F-l028 substance has very low toxicity. Since these physiological, chemical and biological properties of the F-l028 substance do not correspond with those of the known numerous antibiotics, the antibiotic, F-l028 substance is confirmed to be a new antibiotic substance.

The F-I028 substance of the invention can be applied effectively to pathogens living on the aboveground portions of plants, pathogens which invade plants from the soil and cause tracheomycosis, seedinfectious pathogens, and soil-infectious pathogens.

As mentioned before, the F-l028 substance of this invention exhibits superior control effect against pathogens which invade rice plants and other crops, and has good affinity for higher plants. With ordinary dosages, it does not give phytotoxicity to crops, and hardly exhibits toxicity on domestic animals and fish. Thus, this substance can be used against plant pathogens as agricultural and horticultural fungicide quite conveniently, and is practically very valuable for increasing agricultural productivity.

In the application of the F-1028 substance of this invention as an agricultural and horticultural fungicide, it can be used after dilution with water, or as various formulations prepared by the method usually practised in the field of producing agricultural chemicals using agricultural chemical assistants. These various formulations may be applied as such or after dilution with water to the desired concentrations.

As the agricultural and horiticultural assistants referred to herein, there can be named inert solvents and- /or diluents (extenders or carriers) (these are used to carry the active ingredient to pathogenic fungi and/or the habitat of the pathogenic fungi). There can also be cited various surface active agents and/or organic materials, such as stabilizers, spreaders (stickers), propellants for aerosols, or synergizing agents (these are used to produce, maintain, and promote the effect of the active compound to a greater extent).

Examples of the solvent include water, and organic solvents, for example, aliphatic or alicyclic hydrocarbons such as n-hexane, industrial gasolines (petroleum ether, solvent naphtha, etc.), petroleum fractions (paraffin wax, kerosene, light oil, middle oil, heavy oil, etc. aromatic hydrocarbons such as benzene, toluene, xylenes, or aromatic naphtha, halogenated hydrocarbons such as chloromethylene, chloroethylene, carbon tetrachloride, trichloroethylene, ethylene dibromide, or chlorobenzene, alcohols such as methyl alcohol, ethyl alcohol, propyl alcohol, or ethylene glycol, ethers such as ethyl ether, ethylene oxide, or dioxane, alcohol ethers such as ethylene glycol or monomethyl ether, ketones such as acetone and isophorone, esters such as ethyl acetate or amyl acetate, amides such as dimethyl formamide or dimethyl acetamide, and sulfoxides such as dimethyl sulfoxide.

Examples of the diluents (extenders or carriers) include vegetable powders, mineral powders, clay minerals such as kaolinites, montmorillonites or attapulgites, talc, pyrophyllite, mica, gypsum, calcite, muscovite, vermiculite, dolomite, apatite, slaked lime, magnesium lime, diatomaceous earth, inorganic acid salts such as calcium carbonate, sulfur, pumice, synthetic mineral powders such as highly dispersed silicic acid or synthetic alumina, synthetic resins such as phenolic resins, urea resins, or vinyl chloride resins.

Examples of the surface active agents include anionic surface active agents such as alkylsulfuric acid esters (for example, sodium laurylsulfate), arylsulfonic acids (for example, alkylarylsulfonic acid salts or sodium alkylnaphthalenesulfonate), cationic surface active agents such as alkylamines (for example, laurylamine, stearyl trimethyl ammonium chloride, or alkyl dimethylbenzylammonium chlorides), or polyoxyethylene alkyl amines, non-ionic surface active agents such as polyoxyethylene glycol ethers (for example, polyoxyethylene alkylaryl ethers, or polyoxyethylene alkyphenyl ethers), polyhydric alcohol esters (for example, polyoxyethylene sorbitan monolaurate), or polyoxyethylene glycol esters (for example, polyoxyethylene fatty acid esters), and amphoteric surface active agents.

As the examples of the organic materials, there can be cited stabilizers, spreaders (stickers) such as agricultural soap, coumarone or indene resins, or polyvinyl butyl ether, propellants for aerosols such as halogenated hydrocarbons (Freon, etc.), combustion agents for fumigants, such as nitrous acid salts, zinc powder, or dicyandiamide, oxygen-yielding agents such as perchlorates or bichromates, phytotoxicity-reducing agents such as zinc sulfate, ferrous chloride, or copper nitrate, effect-extending agents such as chlorinated terphenyl, dispersion stabilizers such as casein, tragacanth, carboxymethyl cellulose, or polyvinyl alcohol, and synergizing agents.

The compound of this invention can be prepared into various formulations by the method generally practised in the field of producing agricultural chemicals.

Examples of the formulations are liquid preparations, such as emulsifiable concentrates, wettable powders, tablets, soluble powders, or solutions, dusts, granules, fumigants, or aerosols.

The fungicide of this invention may contain the above active ingredient in an amount of 0.1 to 95% by weight, preferably 0.5 to by weight.

In actual application, the suitable amount of the active ingredient contained in the above-mentioned various formulations or in a ready-to-use preparation is generally from 0.0001 to 20% by weight, preferably from 0.005 to" 10% by weight.

The content of the active ingredient may be changed properly according to the formulations, the method, purpose, time, and place of application, and the state of disease occurrence, etc.

If desired, the compound of this invention may further contain other agricultural chemicals such as insecticides, fungicides, acaricides, nematocides, antivirus agents, herbicides, plant growth modifiers, or attractants (for example, organophosphorus esters, carbamates, dithio(or thiol) carbamates, organic chlorides, dinitro compounds, organic sulfur or metal compounds, antibiotics, substituted diphenyl ether compounds, urea compounds, or triazine compounds), and- /or fertilizers.

Various formulations or ready-to-use preparations containing the active ingredient of this invention described above can be applied by the method generally practised in the field of agricultural chemicals, such as spraying (for example, liquid preparation spraying, misting, atomizing, dust spraying, granule spraying, application to the water surface, or pouring), fumigating, application to the soil (for example, sprinkling, vaporing, or pouring), application to the surface of the soil (for example,'coating, banding, dust application, or covering), or impregnation. They can also be applied by the ultra-low-volume spraying method. According to this method, the active ingredient can be included in an amount of up to' or even The amount of application per unit area of the fungicide of this invention is about 3-1000 g, preferably 30-600 g, per 10 ares calculated as the active compound. But in special cases, the amount may either be above or below this range, or sometimes, it is even necessary to deviate from this range.

According to this invention, there is provided a fungicidal composition comprising the compound of formula (l) as an 'active ingredient, and a solvent and/or a diluent (extender or carrier) and/or a surface active agent, and if desired, an organic material.

Furthermore, the present invention provides a method of controlling pathogenic fungi, which comprises applying the compound of formula (I) singly or in admixture with a solvent and/or a diluent (extender or carrier) and/or a surface active agent, and if desired, further with an organic material to said fungi and/or their habitat.

The present invention will be specifically described by the following Examples, but it should be understood that the invention is not limited in any way to these Examples.

FIG. 1 shows a UV spectrum curve of an aqueous solution of the hydrochloride of the antibiotic, the F- 1028 substance, of this invention.

FIG. 2 shows a IR spectrum curve of the hydrochloride of the F-l028 substance mixed with potassium bromide.

EXAMPLE 1 A loopful micelium of Streptomyces kagawaensis, the F-l028 substance-producing strain, was inoculated to a cultural medium containing meat extract 0.5 sodium chloride 0.5 yeast extract 0.3 glucose 2 peptone 0.5 and calcium carbonate. After the inoculated medium was cultivated with shaking for 2 days, it was placed in a 100 lit.-fermentation tank with 75 lit. of the same culture medium. Then this inoculated me dium was incubated at 27C with aeration of lit./- min, agitation of 250 r.p.m., and internal pressure of 0.5 kg/cm for 2 days. The production of the F-l028 substance then reached 400 mcg/ml. in the broth. 70 lit. of the cultivated medium was adjusted to pH 2 by a saturated solution of oxalic acid and filtered with celite at 0.5 Then the filtrate was adjusted to pH 5 with 2N-NaOI-l and stirred together with 2.8 lit. of cation exchange resin, Amberlite IRC-50(Na for 30 minutes. Successively, the resins were collected by filtration, and'the filtrate was adjusted again to pH 5, admixed with 1.4 lit. of Amberlite lRC-50(Na stirred and filtered; These resins were collected, too. By two of these operations, 95 of the F-l028 substance in the filtrate was adsorbed by the resin. Thereafter, the resin which adsorbed the F-1028 substance was washed sufficiently with water and charged into a column. The F-lO28 substance was eluted with lN-HCl, and about 5 lit. of the eluate containing the active substance was collected and concentrated to 100 ml under reduced pressure. After the filtration of sodium chloride formed in this step, three-fold quantity of methanol was added to the solution. It was allowed to stand under cold conditions. The sodium chloride further formed was filtered away, ten-fold quantity of acetone was added. Then the precipitate of the F-l028 substance was formed and 95 g of the F-l028 substance as crude power (Yield: 60 to 70 was obtained by drying the precipitate.

EXAMPLE 2 125 ml of a medium containing glucose 2 soybean meal 3 sodium chloride 0.5 and calcium carbonate 0.5 were placed in a 500 ml-Sakaguchis flask sterilized and inoculated by 2 ml of precultivated medium of Streptomyces kagawaensis, the F-l028 substance-producing strain. Then the flasks were incubated at 27C with shaking at 120 r.p.m. and with 8 cm amplitude. After 48 hours, potency of the F-l028 substance became 580 mcg/ml. The broth was collected and the active compound was extracted by the same methods as in Example 1 to be a crude powder.

EXAMPLE 3 A mutant of Streptomyces kagawaensis, obtained by treatment with N-methyl-N'-nitro-N-nitrosoguanidine and grew with pink or brownish gray aerial mycelia, was preculturcd. And the cultivated medium was inoculated by 2 ml in 500 ml-Sakaguchis flask placed with 125 ml of cultural medium containing glucose 2 peptone 0.5 meat extract 0.5 dried yeast 0.3 sodium chloride 0.5 calcium carbonate 0.3 Then the flasks were incubated at 27C with shaking at r.p.m. with 8 cm amplitude. By the same procedure as in Example 1, the active compound was extracted to obtain the F-l028 substance in a crude powder. The results were shown in Table 4.

6 g of the crude powdered F-1028 substance, obtained in Example 1 was dissolved in 10 ml of water, adsorbed on a column filled with 60 g of activated carbon for chromatography (produced by Wako Seiyaku Co., Ltd.) and developed with water, and the F-l028 substance was eluted in a colorless aqueous solution (Yield: 91.5%). Then the solution was concentrated in vacuo and admixed with about 20-fold quantity of ethanol. The precipitate was dried to obtain 4 g of the crude F-l028 substance in powder. Successively, 2 g of the powder was dissolved in 4 ml of water, poured into Sephadex G-10 column (2.5 X cm) and developed with water. The active fraction was concentrated in vacuo to obtain 1.15 g of white powder. A methanol solution of the white powder was allowed to stand to precipitate crystals. mg of plate-like or needle-like crystals of the F-l028 substance were obtained as hydrochloride.

EXAMPLE 5 An aqueous solution of the hydrochloride of the F- 1028 substance, eluted from the activated carbon column by the procedure as described in Example 4, was passed through a resin column filled with IR-4B[OH The solution passed through the column was neutralized with sulfuric acid, concentrated and admixed with ten-fold quantity of ethanol or acetone. There was obtained a sulfate of the F-1028 substance in powder. The powder was then applied to Sephadex G-lO column to be separated into two fractions. The former was the sulfate, and the latter was the hydrochloride. The latter was again passed through IR4B[Ol-l column. The so lution passed through the column was neutralized with sulfuric acid to obtain the sulfate. An aqueous solution of the sulfate was concentrated to dryness in vacuo to form a white powder.

The excellent features and marked effects of the active compound of this invention, as an agricultural and horticultural fungicide, can be recognized from the results of the following tests in which it was used against various pathogenic fungi.

EXAMPLE 6 Test on sclerotinia rot of beans and gray mold on vegetables (preventive test):

The hydrochloride of F-l028 substance of a prescribed concentration was sprayed to bean (variety: Taisho Kintoki) in the 2-3 leaf stage at a rate of cc per pot. One day after the spraying, the leaves were cut off, and the hypha growing portion of gray mold fungus and sclerotinia rot fungus which had previously been cultivated in a PDA plate culture medium was punched out in a size of 7 mm in diameter and 2 mm in thickness, and inoculated thereto. The inoculated leaves were placed in a constant-temperature vessel (at C., humidity of more than 98%), and after a lapse of three days, the diameters of the enlarged disease spots were measured. Then, the disease spot inhibiting rate was calculated. The results are shown in Table 5 below.

The disease spot inhibiting rate was-calculated as follows: 1

( Diameterof disease spot of untreated leaf dry in air, and again were placed in a constant temperature vessel at 20C. After a lapse of 48 hours, the diameters of the disease spots were calculated. The degree of progress of the disease spots as against the disease spots before spraying of the chemical was expressed by the disease spot inhibiting rate as follows:

Disease spot inhibiting rate (70) (dd-11 r2 41.)

Diameter of disease spot of treated leaf Diameter of disease spot of untreated leaf Table 5 v 1 Sclerotinia Q Gray mold fungus .rot fungus Concent- Diameter lnhibi- Diameter Inhibiratio of of disease ting of disease ting active spot rate spot rate Compound ingredient (mm) (70) (mm) (70) F-l028 substance, 50 ppm 10 67 0 100 hydrochloride ppm 23 23 10 7l F4028 substance, Original ll 63 I00 culture liquor liquid v 8 X I8 40 0 100 Allisan (50%) X i000 (commercially available. control) (500 ppm) l() 67 3 91 Polyoxyne (5%) X 1000 (commercially available, control) (50 ppm) 19 37 2O 57 Non-treated 0 O Allisan: 2.fi-dichloro-4-nitruanilinc.

EXAMPLE 7 dq: diameter of the disease spot of non-treated leaf before application of the chemical dc z diameter of the disease spot of non-treated leaf after application of the chemical dt,: diameter of the disease spot before application of the chemical dt diameter of the disease spot after application of the chemical The results obtainedare shown in Table 6.

Table 6 (A) Test results regarding gray mold Diameter of Diameter of available, control) Non-treated Table o-Continued Diameter of the disease the disease Concentration spot before spot after Disease spot of active spraying spraying inhibiting Compound ingredient (mm) (mm) rate (71 F-1028 Substance I00 ppm 1 1.3 l6.3 76

50 l l.3 19.6 62 Allisan (50%) X I000 13.6 26.6 62 (commercially (500 ppm) available, control) Non-treated 12.0 33 0 Sclex: 3-(3,5-dichlorophcnyl)-5.5-dimethyloxazolidinedione-2.4.

EXAMPLE 8 or an acid salt thereof,

Test on Brown rot (Sclerorinia cineria) of peach (field test):

An aqueous solution of the F-l028 substance with a I fixed concentration was sprayed twice with a 7-day intcrval to peach trees (variety: Kurakata early-ripening, 5th year stage). Both the morbidity upon harvest and morbidity of the fruit packed in a cardbord box for storage at room temperature were investigated. The results are shown in the following table 7.

Table 7 Test results against Brown rot Morbidity after Spraying Morbidity 7 days since Sample compound concentration upon harvest packaging in F4028 substance ppm 0 0 BENLATE (50%) X 1000 0.3 46.l (commercial, control) SCLEX (20%) X l500 0.03 7.7 (commercial, control) Untreated plot 38.5 61.6

Remarks: BENLATE: methyl-l4 butyl carbamoyl)-2-benzimidazole carbamate. SCLEX: 3-(3.5dichlorophenyl)-5,5-dimethyloxazolidine (Hone-2.4.

I claim: 1. A process for the production of the antifungal antibiotic substance F-l028 having the formula:

3. The process for the production of the antifungal antibiotic substance F-1028 of claim 1, in which the pH value of the culture medium is ranging from 6 to 9.

4. The process for the production of the antifungal antibiotic substance F-l028 of claim 1, in which the pH value of the culture medium is 6.2 to 8.5.

5. The process for the production of the antifungal antibiotic substance F-1028 of claim 1, wherein the collection is carried out by adsorbing the F-l028 substance in the cultivated medium with an adsorbant and then eluting out the F- 1028 substance from the adsorbant. 

1. A PROCESS FOR THE PRODUCTION OF THE ANTIFUNGAL ANTIBIOTIC SUBSTANCE F-1028 HAVING THE FORMULA:
 2. The process for the production of the antifungal antibiotic substance F-1028 of claim 1, in which the cultivation is carried out at a temperature ranging from 25* to 35*C.
 3. The process for the production of the antifungal antibiotic substance F-1028 of claim 1, in which the pH value of the culture medium is ranging from 6 to
 9. 4. The process for the production of the antifungal antibiotic substance F-1028 of claim 1, in which the pH value of the culture medium is 6.2 to 8.5.
 5. The process for the production of the antifungal antibiotic substance F-1028 of claim 1, wherein the collection is carried out by adsorbing the F-1028 substance in the cultivated medium with an adsorbant and then eluting out the F-1028 substance from the adsorbant. 